Methods for culturing mammalian taste cells

ABSTRACT

The invention provides methods of culturing mammalian taste cells, including taste receptor cells. Cells are maintained for a duration of up to three months and longer while maintaining molecular and functional characteristics of mature taste cells. The cells are cultured on coated cell culture vessels and, from first replacement of medium onwards, the medium is replaced in intervals of at least 5 days. The invention further provides isolation and culturing methods of taste cells wherein the time that the cells are exposed to isolation solution and proteolytic enzymes is minimized and the cells are cultured in coated culture vessels with the medium replaced in intervals of at least 5 days from first replacement onwards. The invention further provides cultured taste cells, transfection and assay methods, and taste cell assay buffers with an osmolarity of about 300-320 and pH of about 7.0-7.3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/348,274, filed Jan. 2, 2009, now U.S. Pat. No. 8,030,068, whichis a continuation of U.S. application Ser. No. 11/249,854, filed Oct.13, 2005, now U.S. Pat. No. 7,488,599, which in turn claims benefit ofU.S. Provisional Application Ser. No. 60/636,377, filed Oct. 15, 2004.Each of these applications is incorporated herein in its entirety.

TECHNICAL FIELD

The invention is concerned with methods for the isolation and long-termcell culture of mammalian taste cells. Mammalian taste cells includemammalian taste receptor-expressing cells that respond to taste stimulior tastants. The methods of the invention allow the study of tastereceptor cells for an extended period of time, for example up to threemonths or longer. Additionally, the methods of the invention reduce thenumber of animals from which taste cells need to be isolated compared toknown methods. Since the methods of the invention allow taste cells toproliferate and differentiate in vitro, in vitro study of taste cellprocesses including proliferation and differentiation, for example thedetermination of trophic factors required by these processes, is madepossible. The ability to culture taste cells for an extended periodenables experiments requiring a longer timeframe.

BACKGROUND

The taste bud consists of approximately 50-100 taste cells which includethree morphologic types that exhibit both neuronal and epithelialproperties. Based on immunocytochemical characteristics, these tastecells can be classified as type I (dark), type II (light), and type III(intermediate) (Yee et al., J Comp Neurol. 2001 Nov. 5; 440 (1):97-108;Takeda et al., J Comp Neurol. 2004 Nov. 1; 479 (1):94-102).

Studies indicate that approximately 10% of these taste cells exhibitimmunoreactivity for neural cell adhesion molecule (NCAM). WhileNCAM-immunoreactive cells are type III cells, not all type III tastecells are immunoreactive for NCAM (Nelson and Finger 1993, J. CompNeurol 336 (4):507-16), and NCAM expression in taste cells is dependentupon innervation by the IX nerve (Smith et al. 1994, J. Comp Neurol 347(2):187-96). These and other data suggest

that the NCAM-expressing taste cells communicate with nerves.

In contrast, key molecules required for functional responses to tastestimuli are not expressed in NCAM-immunoreactive cells. For instance,gustducin, is a key G-protein involved in taste transduction and is onlypresent in type II cells. However, gustducin is not detected inNCAM-expressing cells. (Takeda et al. 1992, J. Electron Microsc. 41(5):375-80; Yang et al. 2000, J. Comp Neural 425 (1):139-51).

Taste cells are believed to originate from the epithelial cell lineagewith a limited average life span of 10 days, with dying cells beingreplaced by the basal cell population. The large majority of primarycell cultures of taste cells are reported to last a few days at best,e.g. 3 to 5 days maximum (compare, for example, for mouse taste cells amethod modified from the isolation of mammalian central neurons bySpielman et al. 1989, Brain Research 503: 326-29; and for rat tastecells Kishi et al. 2001, Neuroscience 106 (1): 217-25, and Stone et al.2002, Chem. Senses 27: 779-87).

To be able to detect a given tastant, taste cells typically need boththe relevant taste receptor for the tastant (e.g., bitter, sweet, umami)and the molecules necessary for taste transduction. Taste receptors mayinclude one or more of T2Rs and/or one or more of T1Rs. Signaltransduction molecules may include gustducin and phospholipase C. Cellsthat express at least one taste receptor and are able to respond to atleast one taste stimulus/tastant are referred to herein as “tastereceptor cells.” Taste cells thus comprise taste receptor cells amongother cell types.

To date, there is no cell culture model for taste receptor cells and anyin vitro research has had to rely on primary cell cultures of tastecells that are maintained for a limited time. Ookura et al. (2002, InVitro Cell. Dev. Biol.-Animal 38: 365-72) describe a particular type ofcells isolated from mouse taste epithelium that have been sorted basedon their integrin 131 marker and that express the NCAM marker. Thus,this integrin-positive continuous mouse cell culture does not generatecells similar to those responsible for the primary detection of tastestimuli.

Ruiz et al. (2001, Chem. Senses 26 (7):861-73) report the maintenance ofprimary cell cultures derived from taste buds for up to 14 days,provided the cells are kept at room temperature, which is believed toslow down various cellular processes. Notably, cells kept at 37° C.could be maintained only for a few days, as had been previouslyreported. The cells kept at room temperature are reported to start dyingat around day 10, which corresponds to the expected average life span oftaste cells. A protocol published by Ruiz et al. 1995 (“Tissue Cultureof Rat Taste Buds”, Eds. Spielman A I, Brand J G EXPERIMENTAL CELLBIOLOGY OF TASTE AND OLFACTION, CRC Press, 1995) discloses a similarmethod at room temperature that mentions a culture duration of up to 18days.

SUMMARY

The invention is generally directed to methods of isolation, culture,and/or assay of mammalian taste cells, including taste receptor cells,and to cultured mammalian taste cells. Encompassed within the inventionare methods of culturing mammalian taste cells by culturing taste cellsin an appropriate cell culture medium and on (wherein the term “on” inthe context of a culture vessel includes therein) a coated cell culturevessel and, from first replacement of medium onwards, replacing themedium at intervals of at least about 5 days. In some embodiments, thetaste cells comprise taste receptor cells. In some embodiments, thetaste cell comprise only taste receptor cells. In preferred embodiments,the cell culture medium comprises Iscove's medium comprising 15-20% MCDB153, 10% FBS, 10 ng/ml insulin, and antibiotics. The coating of theculture vessel preferably comprises collagen.

Also encompassed by the invention are methods of isolating and culturingmammalian taste cells by isolating tongue epithelium wherein length ofexposure of taste cells in the tongue epithelium to isolation solutionin the presence or absence of proteolytic enzymes is minimized,incubating isolated taste cell epithelium pieces in an appropriate cellculture medium and on a coated cell culture vessel, and, from firstreplacement of medium onwards, replacing the medium at intervals of atleast 5 days. The length of exposure of taste cells to isolationsolution with or without proteolytic enzymes is about 30 minutes orless. In some embodiments, the taste cells comprise taste receptorcells, and in some embodiments, the taste cells comprise only tastereceptor cells. In preferred embodiments, the cell culture mediumcomprises Iscove's medium comprising MCDB 153, FBS, insulin, andantibiotics. In more preferred embodiments, the cell culture mediumcomprises Iscove's medium comprising 15-20% MCDB 153, 10% FBS, 10 ng/mlinsulin, and antibiotics. The coating of the culture vessel preferablycomprises collagen. Further contemplated by the invention are tastecells cultured or isolated and cultured according to the inventivemethods. In preferred embodiments, the taste cell is a taste receptorcell. The taste receptor cell preferably responds to at least one tastestimulus. The cultured taste cells of the invention may be cultured forat least about 10 days at about 37° C. and at least about 20 days atabout 18-37° C. The invention also encompasses cultures of the tastecells. In some preferred embodiments, the invention provides mammaliantaste receptor cells that divide in cell culture for more than 48 hours,preferably for more than about 5 days, and more preferably for more thanabout 2 weeks.

The invention also provides a taste cell assay buffer having anosmolarity of about 300-320 milliosmol and a pH of about 7.0 to about7.3 and methods of maintaining taste cells in such a buffer.

Transfected cultured mammalian taste cells are further contemplated. Thetransfected cells of the invention may be transfected with vector DNA,such as plasmid or viral vector DNA. Also provided are methods oftransfecting cultured taste cells by contacting cultured mammalian tastecells with nucleic acids, such as but not limited to, vector DNA, forexample, viral vector DNA or plasmid vector DNA.

Assay methods using the cultured mammalian taste cells are alsoencompassed by the invention. In some preferred embodiments, the tastecell assay buffer used in the assay methods has an osmolarity of 300-320milliosmol and a pH of 7.0 to 7.3.

Methods for assessing taste response to a candidate taste stimulus byexposing a cultured taste cell to the candidate taste stimulus, andcomparing one or more cellular responses of the taste cell to thecandidate taste stimulus with responses of a cultured taste cell to astandard taste stimulus, wherein the same cellular response by acultured taste cell to the candidate taste stimulus as to the standardtaste stimulus indicates that the candidate taste stimulus causes thesame taste response in taste cells as the standard taste stimulus, areprovided. Also provided are methods for assessing taste response of acandidate taste cell comprising exposing a candidate cultured taste cellto a known taste stimulus, and comparing the cellular response of thecandidate cultured taste cell to the cellular response of a standardtaste cell to the known taste stimulus, wherein the same cellularresponse of the candidate taste cell as that of the standard taste cellto the known taste stimulus indicates that the candidate taste cellshares the response of the standard taste cell to the taste stimulus.

Further provided herein are methods for identifying a taste modifier byexposing a cultured taste cell to the candidate taste modifier in thepresence of a known taste stimulus, and comparing one or more cellularresponses of the taste cell to the candidate taste modifier to thecellular responses in the absence of the candidate modifier, wherein achange in the cellular responses to the stimulus in the presence of thecandidate modifier is indicative of a taste modifier.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Provided herein are isolation and culture methods that provide for theculture of taste cells, including taste receptor cells, for up to 1, 2,or 3 months, or longer. The methods of the invention retain a highpercentage of viable cells, even at a temperature of 37° C. andconsequently without slowing down all cellular processes.

The taste cell isolation and/or culture methods of the inventionpreferably include at least one of the following steps:

-   -   During isolation, the time that the taste cells are exposed to        isolation solution, both with and without enzyme(s), is        minimized. For example, taste cells are preferably not exposed        to isolation solution for longer than about 30 minutes,        preferably not longer than about 20 minutes, and most preferably        not longer than about 15 minutes. For example, taste cells are        preferably exposed to isolation solution employed as described        below with pronase E and elastase not longer than about 30        minutes, preferably not longer than about 20 minutes, and most        preferably not longer than about 15 minutes.    -   The cell culture vessel is coated with an appropriate coating.        An appropriate coating for purposes of the invention is a        coating that produces cell adhesion and proliferation. The        coating preferably comprises collagen. The coating preferably        docs not comprise poly-D-lysine, CELL-TAK™, or MATRIGEL™.    -   An appropriate culture medium is used. An appropriate culture        medium for purposes of the invention is a culture medium that        supports cell viability, proliferation, and differentiation. A        preferable culture medium for the methods of the invention        comprises Iscove's Medium, MCDB 153, FBS, insulin, and        antibiotics. An example of an appropriate culture medium is        Iscove's Medium with 15-20% MCDB 153, 10% FBS, 10 ng/ml Insulin        and antibiotics. The culture medium preferably is not selected        from DMEM, F12 or MCDB153, either alone or with fetal bovine        serum (FBS) and antibiotic supplements.    -   Subsequent to the first replacement of medium (e.g., subsequent        to the first replacement of culture medium at 24 to 48 hours),        the medium should not be replaced at intervals shorter than        about 5 days, preferably not less than about 7 days, and most        preferably not less than about 8 days.        The methods of the invention preferably comprise two or more of        these steps, more preferably three of more of these steps, and        most preferably comprise all four of the steps.

Taste cell culture methods comprising one or more of these steps willprovide a viability of taste cells of at least about 40%, preferably atleast about 60%, most preferably at least about 75% after a cultureduration of at least about 10 days at about 37° C., or at least about 20days at about 18° C. to about 37° C. The taste cell culture methodspreferably provide a viability of at least about 40%, preferably atleast about 60%, and most preferably at least about 75% after a cultureduration of about 1 to 2 months or longer at temperatures up to about37° C. Cell viability may be tested by the Trypan Blue test describedbelow.

Isolation and culture methods comprising each of the four steps achievea viability of taste cells of at least about 80% to about 90% or higherafter a culture duration of at least about 10 days at about 37° C., orat least about 20 days at about 18° C. to 37° C. A culture methodcomprising each of the four steps preferably achieves a viability of atleast about 80%, preferably about 90%, more preferably about 95%, andmore preferably about 98% or higher after a culture duration of at leastabout one month at temperatures up to about 37° C.

The methods of the invention include methods for isolating and culturingmammalian taste cells comprising:

-   -   i) isolating tongue epithelium wherein the time that the cells        are exposed to the isolation solution with or without enzyme(s)        is minimized,    -   ii) incubating isolated tongue epithelium in an appropriate cell        culture medium in a cell culture vessel coated with an        appropriate coating, and    -   iii) subsequent to the first replacement of culture medium, for        example at about 24-48 hours, replacing the medium at intervals        of at least about 5 days.

In some preferred embodiments, the taste cells comprise taste receptorcells. In preferred embodiments, the taste cells comprise only tastereceptor cells. In some embodiments, the time of exposure of the tastecells to isolation solution with or without enzyme(s) is about 30minutes or less, more preferably about 20 minutes or less, and mostpreferably about 15 minutes or less. In some preferred embodiments, thecoating is collagen. In some embodiments, the interval between culturemedium replacement is at least about 7 days and preferably is at leastabout 8 days.

According to the methods of the invention, it is possible to maintaintaste cells with a viability of at least about 40% for at least about 10days at a temperature of up to about 37° C., or at least about 20 daysat about 18° C. to about 37° C. In some embodiments, viability of atleast about 80%, more preferably at least about 90%, even morepreferably at least about 95%, for at least about 10 days at atemperature of up to about 37° C., or at least about 20 days at about18° C. to about 37° C. is achieved according to the methods of theinvention.

It has been found that taste cells attach particularly well to a coatedsurface. The invention thus also provides methods of culturing mammaliantaste cells comprising

-   -   i) culturing taste cells in an appropriate cell culture medium        in a coated cell culture vessel, and    -   ii) subsequent to the first replacement of culture medium, for        example at about 24-48 hours, replacing the medium at intervals        of at least about 5 days.

Taste cells cultured according to the methods of the invention may beisolated according to methods known in the art. Preferably, taste cellsare isolated according to the methods provided herein. In some preferredembodiments, the taste cells comprise taste receptor cells. In somepreferred embodiments, the coating is collagen. In some embodiments, theinterval between culture medium replacement is at least about 7 days andpreferably is at least about 8 days.

The invention also provides methods of maintaining taste cells for atleast about 6 hours, preferably at least about 12 hours, and morepreferably at least about 24 hours, in an improved assay buffer. Theimproved assay buffer has a defined osmolarity of about 300-320milliosmol, preferably 300-310 milliosmol. The osmolarity of the assaybuffer may be adjusted, for example with 5 M NaCl. A too highosmolarity, for example above about 320 milliosmol, is to be avoided ascells start to die and do not accept assay reagents such as fura-2 AMfor the calcium imaging assay when determining the response of cells totaste stimuli (see, for example, Example 9). The assay buffer has a pHof about 7.0-7.3. A preferred pH for the improved assay buffer is about7.15 to about 7.25. As a basis buffer, any suitable buffer may be used.If necessary, the basis buffer may be adjusted in osmolarity and/or pH.A suitable basis buffer is, for example, modified MHNK ringer's solution(80 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM Na-pyruvate, 20 mMHepes-Na, pH 7.2). The maintenance methods are useful when cells are tobe maintained for assay purposes. None of the prior art methods wereable to maintain taste cells including taste receptor cells in buffersolution for a comparable duration in buffer solution while maintainingresponsiveness to taste stimuli.

Analysis of taste cells cultured according to the present inventionshows that they maintain several functional and molecular propertiesthroughout the culture duration, for example up to about one, two, orthree months, or longer. In particular, cells continue to divide (asshown by BrdU labelling) and to differentiate into cells expressingtaste receptor cell-specific markers, including gustducin, phospholipaseC-beta-2 (PLC-β2), TRPM5, T1R3, and T2R5. Further, taste cells culturedaccording to the methods of the invention maintain the ability to beactivated by taste stimuli. For example, taste cells cultured accordingto the methods of the invention are activated by one or more tastestimulants including Denatonium (Denatonium benzoate), Acesulfame K(6-Methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide potassium salt), MSG(Monosodium glutamate), Cycloheximide(3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide),Glycine, and High K buffer (High K buffer is rich in potassium andcontains a modified Modified MHNK ringer's solution with 5 mM NaCl, 80mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM Na-pyruvate, and 20 mM Hepes-Na, pH7.2 with osmolarity adjusted to 300-310 by 5M NaCl; All other stimulantsare commercially available from Sigma, Saint Louis, Mo., USA) as shownby calcium imaging. Calcium imaging detects the elevation ofintracellular calcium in response to activation by taste stimuli, whichis one of several mechanisms of reaction to taste stimuli (entry ofcalcium from outside or release from internal calcium stores).

The invention also provides taste cells, including taste receptor cells,that continue to divide in cell culture for more than about 48 hours.Depending on culture duration, taste cells may continue to divide formore than about 5 days, preferably more than about 2 weeks, and morepreferably more than about 4 weeks or longer. Notably, prior art methodsprovided taste cell cultures where either cell division continues onlyfor a short period immediately following isolation or slows down so muchthat the cells do not survive but for a few days. Ruiz et al. 2001,supra, report that the addition of BrdU to cells already in culture for48 hours resulted in essentially no labelling by BrdU. Whether cellscontinuously divide in culture can be easily tested using BrdUlabelling, as is well-known in the art, for example after at least 2days of culture. Taste cells according to the invention show a highpercentage of BrdU-labelled cells, for example at least about 30% ofcells. Preferably, at least about 40% of cells, more preferably about50% of cells, even more preferably about 60% of cells, and mostpreferably at least about 70% of cells are BrdU-labelled. Usually, atleast about 60-70% of cells are labelled.

Mammalian Taste Cells

Isolated taste papillae described in the examples are derived fromtongues of rats, but material from other mammals may also be employedaccording to the methods of the present invention. All mammals share ahigh similarity in the organization of taste cells in the taste bud andin their cellular and molecular organization. Accordingly, taste cellsof other mammals may also be isolated and/or cultured long-term with asimilar viability. For example, common research animals that may beemployed are rodents including for example, rat, mouse, hamster, andguinea pig. Other mammals that may be employed include, for example,bovine animals, pig, dog, cat, and primate (e.g., ape, monkey, human).

Methods of Taste Cell Isolation

Provided the incubation times with enzymes and isolation solution areshort, isolation methods for isolating tongue epithelial tissue andtaste buds may be performed as is well known in the art. The originalmethod was described by Spielman et al. 1989, Brain Research 503: 26-29.Various modifications of this method are known, as described for exampleby Kishi et al. 2001, Neuroscience 106 (1): 217-25. Stone et al. 2002,Chem. Senses 27: 779-87, and Ruiz et al. 2001, Chem. Senses 26(7):861-73.

The isolation procedure according to the invention involves incubationperiods for the incubation in isolation solution and the incubation withproteolytic enzymes that are sufficiently short so that the desiredduration of cultivation and cell viability is reached, which may beeasily tested as described herein. Preferably, the duration ofincubation both with isolation solution and proteolytic enzymes is about30 minutes or less, preferably about 20 minutes or less, and morepreferably about 15 minutes or less. The present invention employs asingle isolation solution with an incubation of about 2 to about 1minutes without enzymes, preferably on ice, to which one or moreproteolytic enzymes are added and incubated about 10 to about 15minutes.

Enzymes preferred for use in the isolation methods according to theinvention are proteases that hydrolyze proteins sufficiently to separatetaste cells in a short period of time not longer than about 30 minutesso that cells can be separated without damaging their ability to attach,grow, and differentiate. Suitable hydrolytic enzymes are, for example,pronase E and/or elastase. Preferably, more than one enzyme is used. Forexample, a combination of pronase E and elastase can be used in theisolation methods of the invention. Pronase E is a non-specific proteasemixture from Streptomyces griseus and is commercially available, forexample from Sigma, Saint Louis, Mo., USA. Elastase, for examplepancreatic porcine elastase type I (synonyms: pancreatopeptidase E,Elastase hog pancreas. CAS number 39445-21-1: Enzyme Commission (EC)Number 3.4.21.36), hydrolyzes proteins including elastin with apreferential cleavage site of Ala-Xaa, and is commercially availablefrom Sigma, Saint Louis, Mo., USA (for example 1 mg/ml elastase inaqueous suspension, 4 units mg protein. Product No. E1250).

Alternatively, the enzymes may be cloned and expressed as is well-knownin the art and described for example by Baumstark et al. 1963, Biochim.Biophys. Acta 676; and Maniatis et al., 1982, “Molecular Cloning, Alaboratory Manual”, Cold Spring Harbor Laboratory.

The tongue epithelium is preferably minced following its isolation.Preferably, after the isolated epithelium from the region of the tastepapillae (one or more of circumvallate, foliate, and fungiform papillae)is transferred to the culture vessel with medium, the isolatedepithelium is minced using appropriate tools, for example surgicalblades, to provide a mixture that comprises intact taste buds as well aspartially dissociated taste buds. Primary cell cultures derived fromsuch a mixture tend to provide cell cultures that can be kept over alonger period in time, which may be due to better attachment and/orlonger periods of survival and/or growth.

Methods of Taste Cell Culture

Appropriate culture medium to be employed in the methods of theinvention will support viability, proliferation, and differentiation oftaste cells. Culture medium for the methods of the invention preferablycomprises Iscove's medium, preferably supplemented with one or more ofMCDB 153 medium, FBS, insulin, and antibiotics. A preferred medium forcell culture is for example Iscove's medium, preferably supplementedwith one or more of 15-20% MCDB 153 medium, 5-20% FBS, 10 ng/ml insulin,and antibiotics. A suitable FBS concentration is 10%. A suitablecombination of antibiotics is, for example, penicillin, streptomycin,gentamycin and fungizone. A suitable concentration is 100 U/ml/100μg/ml, penicillin/Streptomycin, 2.5 μg/ml Gentamycin, and 0.5 μg/mlFungizone.

Iscove's medium is a highly enriched synthetic medium modified fromDulbecco's Modified Eagle's medium (DMEM) and contains sodium selenite,additional amino acids and vitamins, sodium pyruvate, HEPES buffer andpotassium nitrate instead of ferric nitrate. Iscove's medium iscommercially available for example as “Iscove's Modification of DMEM”from Cellgro® by Mediatech Inc, Herndon, Va., USA (Product No. 10-016).

MCDB 153 medium, for example with L-Glutamine and 28 mM HEPES, withoutSodium Bicarbonate, is a modification of Ham's nutrient mixture F-12 andis a highly enriched medium designed for serum-free growth and useshormones, growth factors, trace elements or low levels of dialysed fetalbovine serum protein. MCDB 153 is commercially available from Sigma,Saint Louis, Mo., USA (Product No. M 7403).

An appropriate temperature for cell culture is any temperature thatallows for cell growth and cellular processes at the desired rate, forexample about 18° C. to about 37° C., including room temperature (18-22°C.) and about 37° C., for example 35 to 39° C. A preferred temperatureis 37° C. which is physiological temperature and provides for cellularprocesses at their usual rate. When culturing cells for example at about37° C., temperature will change over time somewhat to oscillate around37° C. on average, e.g. ±.0.5 or 1° C. depending on the cell culturesystem, as is apparent to the skilled person.

Cell culture is performed in an appropriate environment with appropriateCO₂ concentration and appropriate humidity, as is apparent to theskilled person. For example, depending on the buffer system of thechosen medium, a CO₂ concentration of about 5% is appropriate. Cells areusually cultured under conditions of high humidity, for example about95%. Cell culture vessels, for example dishes or cover slips, accordingto the invention are coated, preferably with collagen, more preferablytype I collagen, and most preferably rat tail type I collagen.

After the first medium replacement which may occur, for example, about24-48 hours after seeding, any subsequent medium replacement isperformed in intervals not shorter than at least about 5 days,preferably at least about 7 days, and most preferably at least about 8days. Depending on culture conditions, chosen medium, concentration ofantibiotics, and growth rate, after about 10 days, the medium generallyshould be replaced.

Any method known in the art may be used to assess cell viability. Forexample, Trypan Blue staining (Sigma, Saint Louis, Mo., USA) may beemployed. Viability may be tested as follows: Trypan Blue (Sigma, SaintLouis, Mo., USA) is added directly to the cell culture medium of theculture vessel to give a concentration of 0.2% (w/v) Trypan Blue. TrypanBlue stains the cell nuclei of dead cells blue. After 5 to 10 minutes,100 cells are counted under a phase contrast microscope at 10×magnification using an ocular grid. Viability is determined as apercentage (viable cells counted divided by total cells counted,multiplied by 100). The procedure is repeated with 2-3 coverslips andthe average determined. Variation between different coverslips isgenerally low. Cell cultures are at least about 80% viable, morepreferably at least about 80% viable, more preferably at least about 90%viable, more preferably at least about 95%, even more preferably atleast about 98%, and even more preferably at least about 99% viable.

Taste Cell Assays

Methods and taste cells according to the invention may be used in assaymethods known in the art to identify candidate stimuli or compounds thatelicit a response in taste cells, including tastants, taste modifiers(including stimuli that enhance, suppress, stimulate or inhibit taste,or influence a particular taste quality), and other candidate stimuli orcompounds that elicit a particular response or effect in taste cells.For example, these assays may be used to identify and evaluate thetaste, taste quality, taste modifying, growth promoting, inhibiting ortoxic effects of candidates. Further they may be employed to identifycandidates for treating taste loss, targets for drug development, andnutritional factors needed for maintenance of healthy taste cells. Ingeneral, such assays involve the comparison of at least one type ofcellular response (e.g. fluorescent signal relating to calciumconcentration) of a candidate or mixture of candidates of interest to astandard.

A candidate stimulus or compound may be, for example a candidate tastantor a candidate taste modifier in presence of a tastant. A standardstimulus or compound may be a known taste stimulus or tastant of a knowntaste quality, or a stimulus/compound that is able to elicit aparticular effect on taste cells. Such an effect may be a growthpromoting, inhibiting, or toxic effect, or the loss of response to tastestimuli, the maintenance of cell growth or health of taste cells. Thecandidate and the standard may be the same (for example a known tastant)if differences in the response of different taste cells, e.g. tastecells of different sources (species, individuals, age, etc.) are to becompared.

Taste cells including taste receptor cells according to the inventionare exposed to the candidate and standard and the cellular response thatresults in a measurable signal is compared. Exposure may be doneconsecutively or in parallel as is well known in the art. Severalstimuli or compounds may be tested together.

Different cellular responses are well known in the art and includechanges in the calcium concentration, pH, and voltage. Changes of theresponse may involve changes in magnitude, latency (defined as the timebetween stimulus exposure and response) or duration. These changes maybe detected by detection methods well-known in the art includingfluorescent compounds sensitive to calcium or pH, voltage sensitive dyes(for example as described by Hayashi et al. 1996, Biophys J., 71(2):1057-70), and electrophysiological recording (for example asdescribed by Ogura et al., J Neurosci. 1997 May 15; 17 (10):3580-7, andZviman M M et al., J Membr Biol. 1996 January: 149 (2):81-8).

There is a large variety of specific assay types and it is apparent tothe skilled person how to set them up in detail. For example, an assaymay be performed as described by Frank M E, “Taste nerve recording inrodents”, In: “Experimental cell biology of taste and olfaction”, Ed byA. Spielman and J. Brand, CRC Press, 1995; Bryant, B P, “Trigeminalnerve recording in rodents”, Ed by A. Spielman and J. Brand, CRC Press,1995; Gilbertson T A, “Patch-clamping of taste cells in hamster andrat”, Ed by A. Spielman and J. Brand. CRC Press, 1995.

An overview of some functional assays is provided herein.

Tastant Identification/Characterization Assay:

Taste cell response to a candidate of unknown taste quality is comparedwith that of a known tastant to identify differences or similarities inthe cellular response. Taste cells are identified by their response to aknown tastant, for example as described in Example 8 or using comparablecellular response assays known to the skilled person. The cellularresponse to the candidate is monitored and compared to the cellularresponse of the same cells to the tastant.

Modifier (Taste Modifier) Assay:

Taste cell responses to a known stimulus (e.g. known tastant) in thepresence and in the absence of a candidate modifier (e.g. tastemodifier) are compared. Cellular responses are detected and compared.Comparison may show that the response is increased, decreased, delayed,prolonged, or unaffected. A candidate modifier producing an increase inthe magnitude of the cellular response to the stimulus or an increase inthe frequency of cells responding to the stimulus shows that thecandidate modifier is useful for enhancing the intensity of the stimulus(e.g. tastant), and may be useful to add to certain products such asfood products, to enhance the given stimulus (e.g. tastestimulus/flavor). A stimulus or compound producing a response of shorterlatency or longer response duration indicates an increased intensity oraltered quality.

Findings of an elevated taste intensity, or a faster or more prolongedtaste perception identify a stimulus or compound as a taste enhancer,that may be added to food products to enhance their taste. A candidatemodifier that results in a decreased magnitude of response, slowerlatency, or shorter response duration to the target stimulus isidentified to be useful as a masking, blocking or suppressing agent ofthe target stimulus. If results show no difference in the response tothe target stimulus in the presence versus absence of the candidatemodifier, the compound is not a modifier for the target stimulus.

Determination of Differences in the Taste Cell Response in DifferentTest Subjects:

The response to a given stimulus (e.g. known tastant, known tastant andtaste modifier) of taste cells derived from different sources iscompared. By employing these assays it can be determined whether thecellular responses to a stimulus or tastant differ between differentsources of taste cells that are selected according to differentcultures, individuals, species or in correlation with differences inage, genetic composition, metabolic or nutritional status, diseasestate, medication use, and therapeutic treatment.

The identified differences allow development of improved taste systemstargeting flavors and flavor compositions, e.g. for food products, whichare tailored to the needs of specific species, populations orindividuals. For example, reduced responsiveness to a particularstimulus in cells derived from elderly versus young subjects wouldidentify a necessary increase/decrease in the stimulus concentration toimprove the taste quality for consumers depending on the age of theconsumer group for which the product is intended. The efficacy of ataste modifier whose activity is known in one species may be identifiedin a different species to identify taste stimuli efficient in thespecies of interest.

Assays Based on Cellular Response of Taste Cells Relating to DifferentCellular Parameters:

Parameters measured include differences in response as detailed above,the frequency of responses to taste stimuli, or specified cellularparameters including proliferation rates of cells, expression, ofprotein markers relevant for taste or taste cell regeneration(proliferation, differentiation, survival), and others as described inthe Examples.

Candidate compounds that influence taste cell regeneration, repair,proliferation, differentiation, survival and replacement, that may beuseful as therapeutic drugs, can be identified by the comparison of thetaste cell response in the presence and absence of a candidate compound.Cells may be exposed to candidate compounds either in vivo or in vitro.

Candidate stimuli or compounds identified by the above-described assaysmay be validated further by one or more of the following methods as iswell known in the art. These include behavioral testing with testsubjects, sensory testing, signal transduction testing, physiologicalmethods (for example nerve recording), as described for example bySpielman. A. I., Brand, J. G. (Editors), “Experimental Cell Biology ofTaste and Olfaction”, CRC Press, Boca Raton, Fla., pp. 437, 1995, andDoty, R. L. (Ed.): Handbook of Olfaction and Gustation, 2nd editionN.Y.: Marcel Dekker, 2003, 1150 pp.

Stimuli or compounds identified by assays employing taste cellsaccording to the invention may be added to food products. “Foodproducts” are meant to include all products applied in the oral cavity,including medicaments, mouthwash and other oral health and hygieneproducts. Further it includes pet food products.

Transfection Methods

The present invention for the first time provides transfected culturedtaste cells. In another of its aspects, the invention is thereforedirected to cultured taste cells, preferably taste cells culturedaccording to the present invention, transfected, for example, by aplasmid vector, and to methods of producing such cells, for example bytransfection, for example, by employing plasmid vectors.

Taste cells, including taste cells and taste receptor cells culturedaccording to the methods of the present invention, may be transfectedusing standard methods for transfection of eukaryotic cells, for exampleas described by Murray, E J, editor (1990), Gene Transfer and ExpressionProtocols Methods in Molecular Biology, Vol. 7, Humana Press, Clifton,N.J.; Perkus M E et al. (1993, J. Tiss. Cult. Meth. 15: 72); Feigner, J.et al. (1993, J. Tiss. Cult. Meth. 15: 63). Alternatively, they may betransfected using the FuGene® 6 Transfection Reagent (Roche Diagnostics,Basel, Switzerland), as shown for an expression vector containing areporter gene in Example 10. Taste cells according to the invention canalso be transfected virally by employing known methods of viraltransfection as is well known in the art. Methods for introducinggenetic material into taste cells using a virus are known (Kishi et al.,2001, Neuroscience 106 (1): 217-25; Stone et al. 2002, Chem. Senses 27:779-87).

Plasmid vector transfection protocols have the advantage thatcomplicated virus handling is avoided. Taste cells according to theinvention can be successfully transfected with good transfectionefficiency with plasmid vectors resulting in fluorescent signals fromtransfected proteins (such as Green Fluorescent Protein) that are easilyvisualized. Using cultured cells according to the invention, a goodtransfection efficiency may be one of at least about 10%, preferably atleast about 20%, more preferably of at least about 50%. Preferably,cells cultivated for at least about 5 days or longer (for example atleast about 7 days, at least about 2 weeks, at least about 3 weeks, atleast about 4 weeks, and at least about 2 months) are used.

The following examples describe several aspects of embodiments of theinvention in greater detail. These examples are provided to furtherillustrate, not to limit, aspects of the invention described herein.

EXAMPLES Example 1 Isolation of Taste Cells Including Taste ReceptorCells from the Rat Taste Papillae (Circumvallate, Foliate) and CellCulture

Employed rats are 1-2 month old adult male Sprague-Dawley rats. Rats aresacrificed by CO₂ inhalation followed by cervical dislocation. Thetongue is dissected proximal to circumvallate papillae and immediatelyplaced into a cold isolation solution (26 mM NaHCO₃, 2.5 mM NaH₂PO₄, 20mM glucose, 65 mM NaCl, 20 mM KCl, and 1 mM EDTA) for 5 to 10 minutes onice.

The preparation is removed from ice and about 1 ml of the isolationsolution is mixed with 1.5 ml/ml pronase E (Protease from Streptomycesgriseus, commercially available from Sigma, St. Louis, Mo., USA, productno. P 6911, with an activity of 5.5 units/mg solid. Unit definition: 1 Uwill hydrolyze casein to produce color equivalent to 1.0 micromole (181microgram) of tyrosine per minute at pH 7.5 at 37° C.) and 1 mg/mlelastase (also called pancreatic porcine pancreas Type I, CAS Number39445-21-1, commercially available from Sigma, Saint Louis, Mo., USA,Prod. No. E1250, aqueous suspension with 4 units/mg protein).

The resulting solution with enzymes is uniformly injected with a 25gauge NORM-JECT® syringe under and around the lingual epithelium ofcircumvallate and foliate papillae of the dissected tongue, which isswollen to about twice its normal size. The dissected tongue with enzymesolution is placed in cold isolation solution and incubated 15 minutesat room temperature.

After the enzyme incubation, the epithelium is gently peeled from theunderlining muscle layer under the dissecting microscope (Stereomaster,Fischer Scientific, Pittsburgh, Pa.) and soaked in isolation solution.The epithelium from the region of the circumvallate and foliate papillaeis isolated and transferred to the culture medium.

The following culture media are employed:

-   -   Iscove's medium (“Iscove's Modification of DMEM” from CELLGRO®        by Mediatech Inc, Herndon, Va., USA, Product No. 10-016)        containing 10% Fetal Bovine Serum (BTI, MA, USA), 15-20% MCDB        153 medium (Sigma, Saint Louis, Mo., USA), 10 ng/ml Insulin and        antibiotics (100 U/ml/100 μg/ml Penicillin/Streptomycin, 2.5        μg/ml Gentamycin and 0.5 μg/ml Fungizone);    -   Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, NY, USA;        Cellgro, USA) with or without 10% FBS;    -   MCDB 153 (Sigma, Saint Louis, Mo., USA).

In the medium, the isolated epithelium from the region of thecircumvallate and foliate papillae is minced into small pieces with thesurgical scissors, so that both whole taste buds and partiallydissociated taste buds are present. The pieces of epithelium are seededonto the selected culture dishes or cover slips. The different culturedishes or cover slips that are employed are:

-   -   rat tail type 1 collagen-coated (3.96 mg/ml diluted 1:4 in        water, BD Sciences, San Diego, Calif.) 18 mm round glass        coverslips (Fisher, USA);    -   Polystyrene culture dishes without coating;

Glass coverslips without coating;

-   -   Glass coverslips coated with matrix gel (2 ml/l, ATCC, USA). The        matrix solution is prepared from the Engelbreth-Holm-Swarm (EHS)        mouse sarcoma (ATCC, USA, Prod. No. CRL-2108). Matrix gel is        employed in three variants as follows: Cells are embedded within        matrix gel before seeding and gently applied on coverslip;        coverslip is coated with matrix gel and cells are seeded on the        polymerized matrix gel; cells are seeded onto matrix gel while        the gel is polymerizing;    -   Glass coverslips coated with poly-D-lysine (Sigma, Saint Louis,        Mo., USA, Prod. No. P-6407, Final working concentration is 0.1        mg/ml).

Before coating or use, coverslips are treated as follows: Incubation in2M NaOH for 1 hour and incubation overnight in 70% nitric acid (HNO₃),followed by incubation for 1 hour in an HCl acid wash. Then thecoverslips are autoclaved in water, rinsed with 70% ethanol and 100%ethanol, and air dried.

The culture dishes or cover slips are incubated at 37° C. in ahumidified environment (95% humidity) containing 5% CO₂. The culturemedium is replaced after 24-48 hours and then every 5-10 days.

Adherence of cells is monitored using a microscope (10×) after overnightculture.

The viability of cells is determined by staining with Trypan Blue(Sigma, Saint Louis, Mo., USA). Trypan Blue is added to the culture dishat a concentration of 0.2% (w/v). After 5 to 10 minutes, 100 cells arecounted under a phase contrast microscope at 10× magnification using anocular grid. Dead cells are recognizable by their blue-stained nucleiunder a phase contrast microscope. An area of the cell culture dish orcoverslip is photographed and the viability of cells (viable cellscounted divided by total cells counted, multiplied by 100) is determinedby counting 100 cells for each of 2-3 coverslips.

Results:

Cells are monitored during the entire culturing period and show thefollowing characteristics when kept using the isolation procedure,collagen-coated culture surfaces and Iscove's culture medium accordingto the invention:

Individual cells and bud type cells are visible 24-48 hours afterplating. Cells grow in form of attached cells and cell clusters for upto day 7-8. After day 5-7, cell clusters which give rise to daughtercells start to detach. Cells maintain their original shape up to day15-20. After day 15-20, part of the cell population starts to changemorphology, though the cells maintain their compact appearancesretaining round cell bodies with or without one or more processes. After20 days, most of the long term cultured cells gain a flatter appearance.Cells maintain a high viability for up to 3 weeks with at least 95%viability, in most dishes at least 98% to 99% viability.

When using another isolation protocol or different cell culture surfaceswithout collagen, cells either do not attach, or if they are culturedthe number of dead cells increases rapidly at 2 weeks at latest.

The percentage of cell attachment is determined after overnight culture.When cells are isolated as described above, seeded on collagen-coatedcoverslips, and cultured in Iscove's medium+20% MCDB 153+10% FBS+10ng/ml insulin+antibiotics (Penn/Strep+Genta+fungizone), cells show acell attachment rate after overnight incubation (12-16 hours) of 15-20%.After 7 days, the cells are found at least 90% viable. After 2 months,the cells are still at least 90% viable.

TABLE 1 Culture dish/cover slip surfaces (cells isolated with theimproved protocol according to the invention and cultured in Iscove'smedium) Primary Cells Viability after Surface/coating attached (%) 20days Polypren/Polstyren without 0% — coating Glass coverslip without 0%— coating Matrix Gel coating on glass 3-5%  — coverslip Poly-D-lysine onglass 0% — coverslip Rat Tail collagen on glass 15-20%     At least 90%coverslip

TABLE 2 Tissue culture media (cells isolated with the improved protocolaccording to the invention and seeded on collagen coated coverslips)Primary Cells Viability of cells at Viability of cells at Tissue culturemedium attached (%) day 14 day 20 or more DMEM 0 — — MCDB 153 0 — —Iscove's Medium  5-10% Viable (at least 90%) 80-90% Iscove's medium +20% 10-15% Viable (at least 98-99%) At day 28: 98-99% MCDB 153 + 10%FBS + At month 2-3: 95% 10 ng/ml insulin + antibiotics (Penn/Strep +Genta + fungizone)

Isolated with the enzymes indicated, on the optimized surface and withthe optimized medium, taste cells are maintained for at least up to 2months, up to 3 months, and potentially longer. The first 3 to 4 weeksthe taste cells maintain a viability near about 98 to 99% as tested withTrypan Blue as described above. After 2 months and at 3 months, thetaste cells still maintain a viability of about 95% and part of thecells proliferate.

Example 2 Proliferation of Taste Cells Including Taste Receptor CellsShown by BrdU Labelling

To determine whether primary cell cultures contain proliferative cells,5-bromo-2-deoxyuridine (BrdU) incorporation is performed as describedbelow for cultures up to 11 days old.

60-70 percent of the taste cells are found labeled with BrdU, whichshows that the taste cells continue to proliferate after a cultureduration of at least up to 11 days when isolated by the protocol ofExample 1 and cultured as detailed below.

Taste cells are isolated from rat as described in Example 1 and seededin collagen-coated coverslips. The cells are cultured for 5-6 days andthen treated with 50 μM BrdU (Sigma, Saint Louis, Mo., USA) dissolved in10 mM DMSO for 24-48 hours, after which BrdU is removed by replacementof the medium with Iscove's medium+20% MCDB 153+10% FBS+10 ng/mlinsulin+antibiotics (Pen/Strep+Genta+fungizone). The cells aremaintained for 3 more days in culture on coverslips and then fixed with4% paraformaldehyde in 0.1 M PB (pH 7.2) for 10 minutes at roomtemperature. After fixation, cells are analyzed for BrdU incorporationby immunohistochemistry. After fixation, coverslips are washed threetimes (5 minutes each) in 0.1 M PBS (pH 7.2), and treated with H₂O₂solution (4 mL phosphate buffered saline (PBS)+0.5 mL 100% methanol+0.5mL 30% H₂O₂) for 20 minutes at room temperature to block endogenousperoxidases. Afterwards the coverslips are rinsed with PBS, thendenatured with 2 N HCl at 37° C. for 30 minutes. After denaturation, thecoverslips are washed with PBS, and incubated with blocking buffer inPBS (SuperBlock™, Pierce Chemical Company, Rockford, Ill.) for 1 hr atroom temperature to reduce nonspecific binding. Then the coverslips areincubated overnight with mouse anti-BrdU (diluted 1:100, Sigma, SaintLouis, Mo., USA B-2531) diluted in 10% SuperBlock with 0.05% TWEEN®20 at4° C. After three washes in PBS, the coverslips are incubated withFITC-conjugated anti-mouse IgG (1:500, Santa Cruz Biotechnology) dilutedin 10% SuperBlock with 0.05% TWEEN®20 for 1 hr at room temperature.Afterwards, coverslips are washed three times in PBS for 5 minutes each,and washed three times for 10 minutes each in water. Finally coverslipsare mounted with a mounting medium (Vectashield®, or Vectashield® withDAPI, Vector Labs, USA).

As a negative control, mouse IgG control sera is used in place of theprimary antibody to exclude non-specific staining of BrdU by theanti-BrdU antibody. As further confirmation, unlabeled cells are stainedwith anti-BrdU antibody. None of these controls indicates any specificstaining related to BrdU and the BrdU staining of the taste cells isfound to be specific.

Example 3 Expression of Taste Cell-Specific Markers by RT-PCR

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis isperformed on taste cells isolated and cultured as described in Example 1according to the fast isolation procedure and cultured oncollagen-coated culture dishes and in Iscove's medium modified asdescribed in Example 1.

The following taste cell specific markers are analyzed by RT-PCR:gustducin, PLC-beta-2 (PLC-β2), TRAMS, T1R3, and T2R5. Beta-actin (ahousekeeping gene) is used as a positive control.

Total RNA is isolated from taste cells cultured for 7-10 days, tastecells cultured for 2 months, and as a positive control, from rat tongueepithelium. RNA is isolated, reverse transcribed and amplified by PCR asdescribed below. In all samples (cultured taste cells 7-10 days, 2months, and rat tongue epithelium), the amplification product of theexpected size is detected. This shows that cultured taste cells continueto express mRNA of taste cell-specific markers even after 2 months ofculture.

Protocol Used for RNA-Isolation, Reverse Transcription and Amplificationby PCR:

Each sample is extracted with TRIZOL™ reagent (Invitrogen Corp, USA) asis well-known in the art, for example according to manufacturer'sinstructions. Alternatively, RNA may be isolated, reverse transcribedand amplified as described by Maniatis et al., 1982, “Molecular Cloning,A laboratory Manual”, Cold Spring Harbor Laboratory.

A total 20 μl volume of 4 μg RNA is reverse transcribed for 90 minutesat 42° C. using the SUPERSCRIPT® First Strand Synthesis System forRT-PCR (Invitrogen Corp., USA). As a negative control to check genomicDNA contamination, samples of RNA are treated in parallel in thepresence and absence of reverse transcriptase and used for PCR. Latertests for genomic DNA contamination by PCR amplification show that thereis no genomic DNA contamination. The known specific primers used to showexpression of gustducin, PLC-β2 beta-actin, T2R3, T1R5, and TRPM5 arelisted below.

Primers are chosen to span one or more introns to exclude confusion withamplified fragments from genomic DNA and ensure the generation of atarget-specific product. Primers for gustducin, PLC-β2, and β-Actin werepublished previously, for example in Rossler et al. 2000, ChemicalSenses 25: 413-421. TRPM5 was published previously by Heiner et al.2003, Biochem. J. 371: 1045-1053. The primer first indicated is theforward primer, the second is the reverse.

Gustducin: (SEQ ID NO: 1) 5′-gat gct agc caa tcc gag aag tag aga gg-3′,(SEQ ID NO: 2) 5′-cgg aga tct gct gtt gaa gag gtg aag ac-3′; PLC-β2:(SEQ ID NO: 3) 5′-ctg gag gct gaa gta aag gag-3′, (SEQ ID NO: 4)5′-gcc cct gca tgt atg tta gg-3′; β-Actin: (SEQ ID NO: 5)5′-tca tgt ttg aga cct tca a-3′, (SEQ ID NO: 6)5′-gtc tt gcg gat gtc cac g-3′; T2R5: (SEQ ID NO: 7)5′-tgg caa atc cac atg aag aa-3′, (SEQ ID NO: 8)5′-gca ggg ata gag gaa tgc aa-3′; T1R3: (SEQ ID NO: 9)5′-gat cag tgg tcc cca gaa aa-3′, (SEQ ID NO: 10)5′-taa gct agc atg gcg aag gt-3′; TRPM5: (SEQ ID NO: 11)5′-caa gat cat cgt ggt aga gc-3′, (SEQ ID NO: 12)5′-tcc aga aca tgt ctg cgt tg-3′.

PCR amplification of cDNA for each RT reaction is performed in a finalvolume of 50 μl containing 2 μl of RT reaction, 1× Ampli Taq Gold® PCRbuffer, 2.5 mM MgCl₂, 1 mM deoxynucleoside triphosphates, 0.4 μM of eachprimer, and 0.25 U/μl of Ampli Tag Gold® polymerases (AppliedBiosystems, Foster City, Calif.). PCR amplification consists of initialdenaturation at 95° C. for 5 min followed by cycles of denaturation at94° C. for 30 sec, annealing at 55° C. for 45 sec, and extension at 72°C. for 30 sec. After 40 cycles of amplification, the final extension isat 72° C. for 10 min. PCR products are separated on 1.4% agarose gelsand stained with 0.2 μg/ml of ethidium bromide to verify their expectedsize.

Example 4 Immunohistochemical Localization of Taste ReceptorCell-Specific Biomarkers Gustducin, PLC-β2 and BrdU

α-gustducin and PLC-β2 are known biomarkers for differentiated tastereceptor cells that respond to taste stimuli. BrdU is a general markerfor cell proliferation. Immunocytochemistry is performed on culturedtaste cells isolated and cultured as described in Example 1 according tothe fast isolation procedure and cultured on collagen-coated culturedishes and in Iscove's medium modified as described in Example 1. Tastecells analyzed are cultured for 7-10 days, or for 2 months, and aretested for their immunoreactivity for α-gustducin, PLC-β2 and BrdU asdescribed in the protocol below. Antibody specificity is confirmed byusing antibody-specific immunoglobulin to detect non-specific binding.Immunostaining with antibody-specific immunoglobulin demonstrates theabsence of non-specific immunostaining

Immunoreactivity for both α-gustducin and PLC-β2 is observed both in7-10 days and in 2 month old cultures, with a similar expressionpattern, i.e. they are expressed in cells of similar morphology.

Protocol for Immunoreactivity:

Cells seeded on collagen-coated coverslips are fixed using 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.2, PB) for 10 minutesat room temperature. After three washing steps (duration 10 minute each)in phosphate-buffered saline (PBS), the coverslips are blocked with 0.3%Triton X-100, 2% normal goat serum, and 1% bovine serum albumin in 0.1 MPBS for 1 hour followed by another three washes in PBS. Primary antibodydiluted in blocking solution is added to the coverslips and incubatedovernight at +4° C. Primary antibodies used were polyclonal rabbitanti-gustducin (dilution 1:500-1:1000) and polyclonal rabbit anti-PLC-β2(dilution 1:1000, Santa Cruz Biotechnology). Coverslips are then washedin PBS and reacted in the dark with ALEXA FLUOR® 488 anti-mouse (1:500,Molecular Probes Inc.) or ALEXA FLUOR® 633 anti-rabbit (1:500, MolecularProbes Inc.) diluted in blocking buffer for 1 hour at room temperature.After the final PBS and water washes, coverslips are mounted withVECTORSHIELD®.

All double immunolabeling studies are done in a similar manner exceptthat the second primary antibody, anti-gustducin or anti-PLC-β2, isintroduced after detection of the first primary antibody anti-BrdU iscompleted. Controls for immunofluorescence microscopy consist ofomitting the primary antiserum and no immunoreactivity is seen underthese conditions.

Example 5 Immunohistochemical Localization of Gustducin, PLC-β2, andBrdU in Parallel

The relationship between cells that have proliferated in vitro and tastecells' specific biomarker expression is analyzed in parallel by doublelabelling with gustducin, PLC-β2, and BrdU. The experiments areperformed as described in Example 4, each with two of the threebiomarkers in parallel.

Taste cells cultured for 7-10 days and 2 months exhibit expression ofspecific markers for differentiated taste cells (gustducin, PLC-β2) aswell as proliferation (the latter is shown by BrdU staining).Experiments are performed by double labelling with gustducin or PLC-β2,each combined with BrdU. While some cells show signals for only one ofeach marker (either BrdU, gustducin, or PLC-β2), the large majority ofcells show signals for two of the markers in parallel (gustducin andBrdU, PLC-β2 and BrdU). This shows that cells have both proliferated anddifferentiated into taste cells in vitro.

Example 6 Confocal Imaging

Fluorescent images are captured with the Leica TCS SP2 Spectral ConfocalMicroscope (LEICA Microsystems Inc., Mannheim, Germany) using UV, Argonand HeNe lasers. The coverslips are viewed under a HC PL APO CS 20.0×(0.070 NA) objective. Excitation wavelengths used are 405 nm for DAPI,488 nm for ALEXA FLUOR® 488, and 633 nm for ALEXA FLUOR® 633 withemissions detected at appropriate wavelengths. The pinhole diameter isset at the first minimum diameter of the Airy disc for the objectiveused, giving acceptable resolution of the z-axis for the fluorescentfocal plane. The power for the laser beam and gain of thephotomultiplier are adjusted to optimize the signal/noise ratio.Sequential acquisition of each wavelength are used for some doublelabelling experiments and showed no signal bleed across the wavelengthor differences when compared with simultaneous scans. LEICA Scanwaresoftware is used to acquire confocal images scanning unidirectionally ata 1024×1024 pixel format with 2 lines plus 3 frames averaging. Computercontrolled digital zoom is used to increase magnification to a maximumof 2.5× under 20× objectives. Digital images are arranged and adjustedfor contrast and brightness using LCS software (LEICA MicrosystemsInc.).

Example 7 Expression of Taste Receptor Cell-Specific Markers Analyzed byWestern Blot

For gustducin and PLC-β2 expression of cultured taste cells, WesternBlots are employed according to the protocol described below.

Cultured taste cells tested are cultured for 1 week and 2 months asdescribed in Example 1 on collagen-coated culture dishes with Iscove'smedium modified according to the invention as described in Example 1.

These cells are tested for their expression of the differentiated tastecell-specific biomarkers gustducin and PLC-β2. As a positive control,samples obtained from freshly isolated rat tongue foliate andcircumvallate tissue lysate are used. Gustducin and PLC-β2 expression isshown in all samples. For PLC-β2, using anti-PLC-β2 antibodies, twodistinct bands are shown that had been previously reported (Wei and Neer2001, J. Biological Chemistry 276, pp. 2503-2508). For both biomarkers,the positive control has a stronger signal and expression level comparedto the cultured cells. Between cells cultured 1 week or 2 months, thereare no differences in signal level. Taste cells isolated and cultured asdescribed in Example 1 maintain their specific biomarkers for at least 2months.

Western blots are conducted using standard immunoblotting techniqueswell-known in the art, for example by Maniatis et al., 1982, “MolecularCloning, A laboratory Manual”, Cold Spring Harbor Laboratory.

Cultured primary rat taste cells (7-10 days and 1-2 months old) arelysed in and tissue samples from rat circumvallate and foliate papillaeare homogenized in RIPA (150 mM NaCl, 10 mM Tris pH 7.2, 0.1% SDS, 1%Triton X-100, 1% Deoxycholate, 5 mM EDTA) buffer containing proteaseinhibitors (104 mM AEBSF, 80 μM Aprotinin, 2 mM Leupeptin, 4 mMBestatin, and 1.5 mM Pepstatin A). The protein concentration isestimated for each sample using Bio-Rad Dc Protein estimation kit(Bio-Rad Laboratories, Hercules, Calif.) as is well-known in the art,for example, according to the manufacturer's protocol. Protein samplesare mixed with SDS loading buffer containing beta-mercaptoethanol,boiled for 5 minutes, and then placed on ice for 5 minutes. The cellularhomogenates are separated by SDS-polyacrylamide (5-15%) gradient gel(Bio-Rad Labs.) electrophoresis and transferred to a PVDF membrane(Bio-Rad Labs) that is incubated at 4° C. overnight with 1% of nonfatdry milk. Polyclonal rabbit anti-gustducin (Antibody gustducin 1-20,Santa Cruz Biotechnology, Santa Cruz, Calif., product No. sc-395)(dilution 1:1000) and polyclonal rabbit anti-PLC-β2 (Santa CruzBiotechnology, Santa Cruz, Calif.) (dilution 1:1000) are used toidentify taste cells. After 1.5 hr incubation with these primaryantibodies at room temperature, the membrane is washed and rinsed 3times by incubating for 15 minutes each in 0.1M phosphate buffersolution with 0.05% TWEEN®20 (“PBS/T”), reacted for 1 hour at roomtemperature with HRP-conjugated secondary anti-rabbit antibody (dilution1:5000, NA 934, Amersham). The membrane is washed and rinsed 3 times byincubating for 15 minutes each in a large volume of PBS/T. Signals aredetected with the enhanced chemiluminescence (ECL) immunoblot detectionsystem (Amersham Biosciences, Piscataway, N.J.) as is well known in theart, for example according to manufacturer's instructions. X-rays filmsare later scanned with a scanner for analysis.

Example 8 Response of Taste Cells to Different Taste Stimuli Determinedby Ca-Imaging

Taste cells employed are isolated as described in Example 1 and culturedfor 1 week or 2 months on rat tail collagen type 1-coated coverslips.

Ca-imaging is performed as follows. The coverslips are incubated for15-30 minutes with calcium sensitive dye in Modified MHNK ringer'ssolution (80 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mMNa-pyruvate, and 20 mM Hepes-Na, pH 7.2 with osmolarity adjusted to300-310 by 5M NaCl) supplemented with 1 mM Fura-2 AM (Molecular ProbesInc., Eugene, Oreg.) and 20 mg/ml PLURONIC® F127 (Molecular ProbesInc.). The calcium sensitive dye fura-2 AM is cleaved to fura-2 which isfluorescent and can be detected.

After incubation, coverslips are placed in a recording chamber andcontinuously bathed with Modified MHNK Ringer's solution that is appliedas superfusion.

The stimuli (Denatonium benzoate 2 mM and 0.5 mM, Acesulfame K 250 ppm,Monosodium glutamate (MSG) 3 mM, Cycloheximide 25 μM, Glycine 125 mM,and High K buffer (modified Modified MHNK ringer's solution with 5 mMNaCl, 80 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM Na-pyruvate, and 20 mMHepes-Na, pH 7.2 with osmolarity adjusted to 300-310 by 5M NaCl) areapplied to the coverslip by switching the superfusion to the stimulussolution, which allows for a complete change of bath solutions in thechamber within 10 seconds.

Calcium imaging recordings are performed using standard imagingtechniques as described by Restrepo D., M. Zviman and N. E. Rawson, “Themeasurement of intracellular calcium in chemosensory cells”, in: Methodsin Chemosensory Research, Ed. by A. Spielman and J. Brand. CRC Press,1995. Illumination is provided by a LSR SPECTRAMASTER monochromatorcoupled to the microscope. Emitted light from fura-2 in the cells under200× magnification is filtered at 510 nm and recorded with a cooled CCDcamera (Olympix, Perkin Elmer Life Sciences, Bethesda Md.). Theexcitation wavelength is 340-380 nm and the emission wavelength is 510nm using a wide band filter. Images are digitalized using a MerlinImaging Workstation (Perkin Elmer Life Sciences, Bethesda Md.), whichcontrols illuminator, camera, and acquisition, and performs the imageratioing and the display of pseudocolor images. After introduction tothe recording setup, cells remain viable for over 2 days and can beimaged continuously for 2 hours at a time without visible effects of dyebleaching.

Stimuli are diluted in Modified MHNK ringer's solution and applied via agravity-flow superfusion apparatus for about 10-60 seconds, depending onthe stimulus. Cells can be stimulated by all stimuli tested as shown bya positive signal for Fura-2 AM for at least one cell selected.Typically several cells are tested.

Example 9 Maintenance of Taste Cells in Improved Buffer for Assays

Cultured taste cells employed are as described in Example 8. Culturedtaste cells on coverslips are incubated overnight in an improved assaybuffer outside of the incubator at room temperature (22-25° C.). Theimproved assay buffer is modified MHNK ringer's solution (80 mM NaCl, 5mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM Na-pyruvate, 20 mM Hepes-Na, pH7.2) with the osmolarity adjusted to 300-310 by 5 M NaCl.

In this assay buffer and under the above incubation conditions, thecells are maintained for up to 24 hours and still respond to the stimuli(compare Example 10 for stimuli applied) as determined by positiveFura-2 AM signal (performed as described in Example 8). Cells show thesame results compared to 24 hours before examination. When exposed tohigher osmolarities of 320 or higher, cells die and do not stain withfura-2 AM. This maintenance of primary taste cell cultures in assaybuffer is useful for assay purposes.

Example 10 Transfection of Cultured Taste Cells

Taste cells isolated as described in Example 1 cultured ontocollagen-coated-coverslips are transfected at day 5-7 after isolationwith 5-8 μg pGFP2-MCS-Rluc (h) expression vector (BioSignal Packard,Montreal, Canada, Product No. 6310051) and FuGene® 6 TransfectionReagent (Roche Diagnostics, Basel, Switzerland) according to the RocheDiagnostics “FuGene 6 Transfection Reagent” Instruction Manual, Version5, September 2000. 15-24 microliters of Fugene 6® Transfection Reagentare added to 50 microliters serum-free medium. The medium that is usedis supplemented Iscove's medium as described in Example 1 except that itis serum-free. The mixture is mixed by hand gently. Five to eightmicrograms DNA (pGFP2-MCS-Rluc (h) expression vector) are added, and themixture is mixed by hand gently. The mixture is incubated at roomtemperature for 15-30 minutes and then added dropwise onto the cells.The plate is swirled gently and incubated at 37° C. in a cell cultureincubator for 2 days.

Alternatively, cells may be transfected using standard methods fortransfection of eukaryotic cells, as is well known in the art, forexample as described by Murray, E J, editor (1990), Gene Transfer andExpression Protocols: Methods in Molecular Biology, Vol. 7, HumanaPress, Clifton, N.J.; Perkus M E et al. (1993, J. Tiss. Cult. Meth. 15:72); Feigner, J. et al. (1993, J. Tiss. Cult. Meth. 15: 63).

After 24-48 hours, cells are fixed in 4% paraformaldehyde (PFA) in PBSfor 10 minutes and fluorescent signals of Green Fluorescent Protein(GFP) are detected by confocal microscopy. Expression of GFP is easilyvisualized.

This shows that taste cells according to the invention provide afeasible expression system in a system more similar to in vivo than thecell lines typically used. Similar to the test-DNA used (pGFP2-MCS-Rluc(h) expression vector), DNA of taste receptors may be over-expressed.

Example 11 Transfection of Cultured Taste Cells with mT2R5 and hT2R16and Calcium Imaging

Transfection in Example 11 is performed as described in Example 10subject to the following modifications:

the coverslips used are rat tail collagen-1-coated coverslips;

transfection is performed at day 3-7 after isolation; and

transfected DNA is the expression vector of Example 10 with GFP or anexpression vector carrying a DNA insert, either mouse T2R5DNA(sst:mT2R5:HSV/pcDNA3.1-zeo, 6053 bp) or alternatively, human T2R16DNAas insert (in both cases, the construct may be formed as described forthe cloning of the human TAS2R genes by Bufe et al. Nat. Genet. 2002November; 32 (3):397-401).

Expression is detected in cultured taste cells using antibodies againsttag protein (Herpes Simplex Virus Glycoprotein D, HSV-D). Transfectedand untransfected cells are incubated overnight at 4° C. with anantibody against gustducin (rabbit polyclonal anti-gustducin, (SantaCruz, 1:1000), washed with PBS (3 times, 15 min. each), and thenincubated for 30 min at room temperature with goat anti-rabbitALEXA®-633 (Molecular Probe, 1:500), washed with PBS (3 times, 15 mineach), and then incubated with the mouse anti-HSV-Tag Monoclonalantibody (Novagen #69171-3, 1:1000) overnight at 4° C., washed with PBS(3 times, 15 min. each), then incubated with goat-anti-mouse ALEXA® 488(Molecular Probe, 1:500) for 30 minutes at room temperature. Coverslipsare then washed three times in PBS for 10 minutes each, and three timesfor 10 minutes each in water, mounted with a mounting medium(Vectashield®, or Vectashield® with DAPI, Vector Labs, USA) and viewedon a confocal or epifluorescence-equipped microscope. Controls fornon-specific immunoreactivity include omitting the primary antibodiesand substitution of the primary antibodies with rabbit and mouse IgG, inwhich case no immunostaining is detected.

Both GFP, and HSV-D tag probes show strong fluorescence signalsindicative of over-expression. The signal for HSV-D tagimmunofluorescence is correlated with the expression of T2R5 or T2R16protein, respectively. These results demonstrate that the cultured tastecells according to the invention provide a feasible expression system.

Alternatively, transfected cells are used for calcium imaging assays asdescribed in Example 8. Cells transfected with T2R5 as described exhibitan increase in intracellular calcium in response to cycloheximide ateffective concentrations of 1 microM cycloheximide, which is comparableto the concentration observed in HEK293 cells transfected with thisreceptor by Ueda et al., J. Neurosci., 2003. 23 (19):7376-7380.

Cells transfected with T2R16 as described exhibit an increase inintracellular calcium in response to phenyl-beta-D-glucopyranoside at aneffective concentration of 1 milliM which is comparable to theconcentration observed in HEK293 cells transfected with this receptor byBufe et al Nat. Genet. 2002 November; 32 (3):397-401.

Again, these results demonstrate that the cultured taste cells accordingto the invention provide a feasible expression system.

It should be appreciated that the present invention is not limited tothe specific embodiments described above, but includes variations,modifications, and equivalent embodiments defined by the followingembodiments. Further, all embodiments disclosed are not necessarily inthe alternative, as various embodiments of the invention may be combinedto provide the desired characteristics.

What is claimed is:
 1. A method of transfecting a mammalian tastereceptor cell comprising: contacting mammalian tongue epithelium tissuewith proteolytic enzymes, thereby yielding enzyme-exposed tongueepithelium tissue; culturing said enzyme-exposed tongue epitheliumtissue on a collagen-coated surface in culture medium, wherein saidculture medium comprises Iscove's medium; replacing culture medium atabout 24 to 48 hours and then every about 5 to 10 days, therebygenerating a mammalian taste receptor cell; and contacting saidmammalian taste receptor cell with a nucleic acid.
 2. The method ofclaim 1 wherein said nucleic acid comprises vector DNA.
 3. The method ofclaim 2 wherein said vector DNA comprises viral vector DNA or plasmidvector DNA.